Chytrid Fungi Construct Actin-Rich Pseudopods, Implicating Actin Regulators WASP and SCAR in an Ancient Mode of Cell Motility
Total Page：16
File Type：pdf, Size：1020Kb
bioRxiv preprint doi: https://doi.org/10.1101/051821; this version posted May 4, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Chytrid fungi construct actin-rich pseudopods, implicating actin regulators WASP and SCAR in an ancient mode of cell motility Authors: Lillian K. Fritz-Laylin,a Samuel J. Lord,a and R. Dyche Mullinsa,1 a Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco CA 94158, USA. 1 Corresponding author: Department of Cellular and Molecular Pharmacology University of California, San Francisco Genentech Hall, Room N312, Box 2200 600 16th Street San Francisco, CA 94158 Phone number: 415-502-4838 Email: [email protected] Abstract Various cells scattered throughout the eukaryotic tree crawl across surfaces or through three- dimensional environments. Evidence now indicates that cell crawling is not a single behavior, but rather a collection of processes, driven by different molecular mechanisms. Understanding these mechanisms and their evolutionary relationships first requires narrowly defining mechanical modes of locomotion, and then identifying phenotypic and molecular markers of each. Here, we focus on a widely dispersed form of cell crawling characterized by dynamic, actin-filled pseudopods and weak, nonspecific adhesion to external substrates, a mode we refer to as “α-motility.” Finding α-motility in cells ranging from free-living amoebae to immune cells hints at a single evolutionary origin of this complex process. By mining recently published genomic data, we identified a clear trend: only organisms with both WASP and SCAR/WAVE— two activators of branched actin assembly—make dynamic, three-dimensional pseudopods. While SCAR has been shown in some organisms to be an important driver of pseudopod formation, a role for WASP in this process is less clear. We hypothesize that these genes together represent a genetic signature of α-motility, and both proteins are used for pseudopod formation. We test our hypothesis by depleting WASP from human neutrophils, and confirm that both proteins are needed for explosive actin polymerization, pseudopod formation, and rapid cell migration. We also found that WASP and SCAR colocalize to the same dynamic signaling structures in living cells. Moreover, genomic retention of WASP together with SCAR also correctly predicts the presence of pseudopods in the disease-causing fungus Batrachochytrium dendrobatidis, making it the first fungus reported to undergo α-motility. By narrowing our focus to a single mode of cell migration while expanding our phylogenetic analysis to a variety of eukaryotes, we identified WASP together with SCAR as a conserved genetic marker of fast, low-adhesion cell crawling. Our cell-biology experiments argue that this conservation reflects their collaboration in the explosive actin assembly required to build dynamic pseudopods. These results represent the clearest evidence for a widely distributed mode of cell crawling with a single evolutionary origin. 1 of 23 bioRxiv preprint doi: https://doi.org/10.1101/051821; this version posted May 4, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Eukaryotic cells move using several distinct modes of locomotion, including crawling and flagella-driven swimming. The stereotyped architecture of flagella and the conservation of their protein components render the evolutionary history of cell swimming relatively transparent. In contrast, “crawling motility” is a collection of processes whose functional and evolutionary relationships are not well understood [1-3]. Some crawling cells require dedicated adhesion molecules to make specific, high- affinity contacts with their surroundings, while other cells rely on weaker, nonspecific interactions. Crawling cells also employ different mechanisms to advance their leading edge, either assembling polymerized actin networks to push the plasma membrane forward, or detaching the membrane from the underlying cytoskeleton to form a rapidly expanding bleb. Furthermore, some cell types have been shown to use contractile forces to generate forward movement [4-6]. Different cells can also employ different sets of molecules to drive similar modes of crawling. In an extreme example, nematode sperm have evolved a method of crawling in which polymer assembly advances the leading-edge membrane but, in these cells, the force-generating polymer networks are composed of “major sperm protein” rather than actin. Given this variety of crawling behaviors, it is clear that one cannot simply assume that the underlying molecular mechanisms are the same. The best understood mode of crawling is the slow (1-10 µm/hour) creeping of adherent animal cells, including fibroblasts and epithelial cells [7]. These cells move by extending sheet-like protrusions called lamellipodia while gripping substrate molecules using integrins, often clustered into large focal adhesions. Although clinically and physiologically important, this form of adhesion-based crawling is unique to the animal lineage, and largely restricted to molecular “highways” formed by the extracellular matrix. In contrast, many motile cells—including free-living amoebae and human immune cells—make three-dimensional, actin-filled pseudopods and navigate complex environments at speeds up to 20 µm/min (100-1000× faster than creeping fibroblasts) without forming specific molecular adhesions [8,9]. Although this mode of fast cell crawling has been called “amoeboid motility,” this term is also used to describe a range of behaviors, including cell motility that relies on membrane blebs rather than actin-filled pseudopods [2]. To narrow our mechanistic focus we use the term “α-motility” specifically to describe cell crawling that is characterized by: (i) highly dynamic three- dimensional, actin-filled pseudopods at the leading edge; (ii) fast migration, often on the order of 10 µm/min; and (iii) the absence of specific, high-affinity adhesions to the extracellular environment. Some organisms using α-motility may also employ additional methods of generating forward movement, such as contractility or retrograde flow [4-6], but here we focus on a phenotype that is readily observable in diverse species, including non-model organisms. Organisms with cells capable of this form of locomotion appear throughout the eukaryotic tree, and we hypothesize that α-motility reflects a single, discrete process that arose early in eukaryotic evolution and has since been conserved. If this hypothesis is correct, then elements of this ancient process—specific molecules and mechanisms—should be conserved and still associated with cell crawling in distantly 2 of 23 bioRxiv preprint doi: https://doi.org/10.1101/051821; this version posted May 4, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. related organisms that employ α-motility. Such molecular remnants would help to unravel the evolutionary history of cell locomotion, and might enable us to predict the existence of specific modes of motility in unexplored species. Identifying genes associated with a process such as α-motility is not trivial because the core machinery driving pseudopod formation (e.g. actin and the Arp2/3 complex) is shared with other cellular activities, including some types of endocytosis [10]. We therefore turned our attention to upstream regulators of actin assembly. WASP and SCAR (also known as WAVE) are widely conserved “nucleation promoting factors” that stimulate branched actin network assembly by the Arp2/3 complex in response to various upstream cellular signals [11-13] and promote different levels of Arp2/3 activity [14]. The broad phylogenetic distributions of WASP and SCAR gene families suggest that both genes are ancient and likely to have been present in the eukaryotic ancestor [15,16], making them appealing candidates for genetic markers of α-motility. SCAR plays a major role in the formation of lamellipodia required for slow, adhesion- dependent migration of fibroblasts [17,18] as well as pseudopods at the leading edge of fast-moving amoebae and neutrophils [19,20]. In contrast, the involvement of WASP genes (including mammalian WASP and N-WASP) in cell crawling is less clear. Several studies argue that WASP family proteins do not participate in membrane protrusion or cell migration [21-23], while other studies suggest a direct role for WASP in pseudopod formation and/or cell crawling [24-29]. Part of this confusion may reflect differences in regulation and assembly of substrate-attached lamellipodia by slow-moving cells and three-dimensional pseudopods made by fast-moving cells. Here, we clarify the role of WASP in cell migration by focusing specifically on the regulation of actin assembly in pseudopods used for α-motility. To understand the regulation of the actin cytoskeleton during pseudopod formation, we exploited the diversity of organisms that use α-motility. By comparing the genomes of many eukaryotes, we found that only organisms with genes encoding both WASP and SCAR make pseudopods. We validated this molecular signature using a positive test (a new prediction of α-motility in a little-studied organism) as well as a negative test (depleting